The complex behaviors of all vertebrates are determined by the brain where neurons are connected by synapses. The average volume of synapses corresponds to a sphere of ~400 nm radius?a size scale that can barely be resolved using conventional optical microscopy methods. Synapses are tightly packed with molecular assemblies of synaptic vesicles, synaptic and cytoskeletal proteins and neurotransmitter receptors. These protein complexes are assembled in a much smaller scale?15-60 nm in the three-dimensional tissue space. Current state-of-the-art imaging methods including optical super-resolution microscopy and expansion microscopy remain limited in terms of its achievable resolution in 3D, resolution deterioration in thick tissues, multiplexing capability and the access to the link between functional and structural connectivities. We propose to develop a light-sheet illuminated, adaptive optics assisted, ultra-high resolution (10-15 nm 3D resolution) 4Pi/interferometric single-molecule super-resolution nanoscopy system for thick tissue specimens (Aim 1). We will perform in vitro functional optogenetic neural circuit mapping using automated patch clamp followed by the imaging of the same brain slices using our novel nanoscopy system to autonomously trace and resolve probed circuit (Aim 2). To provide the highly multiplexed in situ protein detection capability (up to 8 targets), we will combine Exchange-PAINT that utilizes DNA-barcode conjugated antibodies with the developed system (Aim 3). Our system will allow comprehensive mapping of ultrastructural features of neuronal circuits (such as pre- and postsynaptic proteins in different types of synapses) in an functionally analyzed brain tissue with 10-15 nm optical resolution in all three dimensions throughout a cortical microcircuit.